This application is being submitted in response to NOT-OD-19-071. In our MPI R01 parent grant ?Epigenetics of Down syndrome?, we are using mouse models to address the mechanisms of altered epigenetic patterning in Down syndrome (DS), and how the epigenetic changes can affect developmental phenotypes. In this supplement/revision application, we propose to study epigenetic and biological aging and the roles of these processes in co-occurring age-related conditions, using our segmental duplication mouse models of DS, namely Dp(10)1Yey & Dp(16)1Yey, which contain segmental duplications of the regions of mouse chromosomes 10 and 16 (Mm10, Mm16) that show conserved synteny with human chromosome 21 (Hsa21). The medically significant phenotypes to be investigated are age-related immune system alterations, age-dependent hearing loss, and age-dependent cognitive decline (independently of Alzheimer?s disease, AD). We believe that using the Dp(10) and Dp(16) mouse models, and Dp(16) mice with App gene dosage normalized to disomy, we can start to use genetic dissection to help us to answer the following questions: (i) what are the genes on Hsa21 that cause early epigenetic aging in DS? (ii) what are the genes on Hsa21 that cause early biological aging in DS (iii) is epigenetic aging simply a useful ?clock?, or do epigenetic changes functionally influence biological aging? and (iv) does early biological aging contribute to the early onset of immune system deficits, hearing loss, and cognitive decline (independently of AD) in DS? We have two specific aims: (1) By methyl-seq & RNA-seq of brain and immune system cells of the above models at defined ages, we will ask whether early epigenetic aging occurs in these segmental models, and whether this phenomenon requires duplication of both chromosomal regions. (2) By analyzing the aforementioned clinical phenotypes in the same mouse models at each of the same ages as in Aim 1, we will determine whether the duplications of Hsa21 syntenic regions on Mmu10 and Mmu16 affect these age-dependent phenotypes. Like DS in humans, App gene triplication is necessary for DS mice to exhibit Alzheimer-type neurodegeneration. By normalizing App gene dosage to two copies in the segmental duplication mice, we will determine the impact of early aging on cognitive decline, independently of AD pathology, by using synaptic density analysis, hippocampal long-term potentiation and behavioral paradigms of learning and memory at successive ages. Attaining these objectives will set the stage for identifying the Hsa21 gene ortholog(s) that cause early epigenetic and biological aging in DS and thereby affect the age-of-onset of immune system alterations, hearing loss, and AD-independent cognitive decline. These results will be relevant both for understanding and ameliorating co-occurring conditions in people with DS and for ameliorating these same problems in the general (euploid) population.